The ribosome is a ribonucleoprotein machine responsible for protein synthesis. In all kingdoms of life it is composed of two subunits, each built on its own ribosomal RNA (rRNA) scaffold. The independent but coordinated functions of the subunits, including their ability to associate at initiation, rotate during elongation, and dissociate after protein release, are an established paradigm of protein synthesis. Furthermore, the bipartite nature of the ribosome is presumed essential for biogenesis since dedicated assembly factors keep immature ribosomal subunits apart and prevent them from translation initiation [Karbstein 2013]. Free exchange of the subunits limits the development of specialized orthogonal genetic systems that could be engineered or evolved for novel functions without interfering with native translation.
The ribosome is an extraordinary complex machine. This large particle, in which RNA is the main structural and functional component, is invariably comprised of two subunits that coordinate distinct but complementary functions: the small subunit decodes the mRNA, while the large subunit catalyzes peptide-bond formation and provides the exit tunnel for the polypeptide. The association of the subunits is tightly regulated throughout the cycle of translation. First, several assembly factors prevent the two subunits from associating during maturation of the ribonucleoproteins. Later on, the initiation of translation is also strictly controlled such that small subunit is involved in the first steps of initiation, while the large subunit is kept apart. Initiation factors, mRNA and fMet-tRNAfMet sequentially join the small subunit to form a pre-initiation complex before recruiting the large subunit. During elongation, the subunits ratchet relative to each other with an angle of about 6 degrees. Upon termination, the newly synthesized protein is released from the ribosome and the subunits dissociate during an active process called ribosome recycling to prepare for subsequent rounds of translation. Thus, the requirement for programmed subunit association and dissociation at specific stages of translation is considered a prerequisite for protein synthesis and likely explains why the ribosome has been maintained as two subunits during the course of evolution. Although initiation at the leaderless mRNAs was suggested to be carried out by the 70S ribosome with pre-associated subunits, no experimental evidence exists showing that the full cycle of protein synthesis could be accomplished by the ribosome with inseparable subunits.
The random exchange of ribosomal subunits between recurrent acts of protein biosynthesis presents an obstacle for making fully orthogonal ribosomes, a task with important implications for both fundamental science and bioengineering. Previously, it was possible to redirect a subpopulation of the small ribosomal subunits from translating indigenous mRNA to translation of a specific mRNA by placing an alternative Shine-Dalgarno (SD) sequence in a reporter mRNA and introducing the complementary changes in the anti-SD region in 16S rRNA [Hui 1987; Rackham 2005], which enabled selection of mutant 30S subunits with new decoding properties [Wang 2007]. However, because large subunits freely exchange between native and orthogonal small subunits, creating a fully orthogonal ribosome has been impossible thereby limiting the engineering of the 50S subunit, including the peptidyl transferase center (PTC) and the nascent peptide exit tunnel, for specialized new properties.
The engineering of a tethered ribosome, in which the subunits are linked to each other, opens new venues preparing orthogonal translation systems, evolving the ribosome for the incorporation of unnatural amino acids in synthetic biology, and molecularly characterizing mutations of functionally critical nucleotides which are often associated with lethal phenotype. Previously, we and others disclosed tethered ribosomes and methods of making and using tethered ribosomes. (See International Published Application WO 2015/184283, “Tethered Ribosomes and Methods of Making and Using Thereof,” and Orelle et al., “Protein synthesis by ribosomes with tethered subunits,” Nature, 6 Aug. 2015, Vol. 524, page 119). Here, with disclose improvements to ribosomes with tethered subunits including ribosomes having tether sequences with improved functionality and orthogonal Shine-Dalgarno/anti Shine-Dalgarno pairs for improved orthogonal performance.